Area of gap junctions and coupling resistance of individual cell pairs will be determined to allow calculation of single channel values. Changes in junctional resistance will be studied to determine if opening and closing of single channels can be detected. Particle arrays at gap junctions will be studied by freeze fracture during formation and after coupling. Fixed and unfixed but cryoprotected cells will be compared. Effects of divalent ions on junctional resistance will be evaluated. Inhibitors of cell movement, e.g., cytochalasin B, will be applied to examine the role of movement in junction formation. Voltage dependence, recently discovered, will be analyzed by a dual voltage clamp system in terms of opening and closing rates and voltage dependence of these rates. Low cytoplasmic pH reduces both junctional conductance and its voltage sensitivity; a common pH and voltage sensitive site is indicated. Presumably low pH also affects opening and closing rates. Determination of the pH will suggest application of appropriate group specific protein reagents which will be used to prove junctional structure and its sensitivity to pH and voltage. Temperature effects on conductance, voltage sensitivity and pH sensitivity will also be investigated. Because junctions can be clamped at intermediate conductances, noise of opening and closing channels should occur. Noise will be measured which will allow independent estimation of opening and closing rates. More important an independent value for single channel conductance should be obtained. The goal is to characterize the channels connecting the cell cytoplasms and to analyze the mechanisms involved in their formation and disruption. Insight should be provided into the role of these junctions in development and also into their functioning in the adult tissues in which they are so prevalent.